1. Field of the Invention
The present invention relates, in general, to polysaccharide-protein conjugates and vaccines. In particular the present invention relates to polysaccharide-protein conjugates that elicit serum IgG and IgM antibodies to poly xcex1(2xe2x86x928) NeuNAc, or to both poly xcex1(2xe2x86x928) NeuNAc and poly xcex1(2xe2x86x929) NeuNAc, or to poly xcex1(2xe2x86x928),xcex1(2xe2x86x929) NeuNAc.
2. Background Information
Neisseriae meningitidis are a major cause of systemic infections, especially meningitis, in humans. Capsular polysaccharide (CP) vaccines are licensed for meningococcal groups A,C,Y and W135. Diseases caused by group B meningococci continue to occur in endemic and epidemic forms and remain an important health problem (Gotschlich, E. C. (1984) in Bacterial Vaccines. Ed. Germanier (Academic Press, NY) pp. 237-255; Peltola, H. (1983) Rev. Infect. Dis. 5, 71-91; Poolman, J. T. et al. (1986) Lancet, ii,555-557). Escherichia coli (E. coli) K1 is a major cause of neonatal meningitis, upper urinary tract infections and systemic infections in hospitalized patients and in domesticated and laboratory animals (Robbins, J. B. et al. (1974) N. Eng. J. Med. 290, 1216-1220; Kaijser, B. et al. (1977) Lancet i, 663-664; Cross, A. S. et al. (1984) J. Infect. Dis. 149, 184-193; Ørskov, I., and Ørskov, F. (1985) J. Hyg. Camb. 95, 551-575). Despite antibiotic treatment and supportive care, meningitis caused by these two pathogens continues to exert a high morbidity, including permanent CNS injury, and mortality (Peltola, H. (1983) Rev. Infect. Dis. 5, 71-91; Schneerson, R. (1988) in Understanding Mental Retardation. ed. Kavanagh, J. F. (Paul Brookes Publishing Co. Baltimore), pp. 237-249; Brandtzaeg, P. et al. (1989) J. Infect. Dis. 159, 195-204; McCracken, G. H., Jr. et al. (1974) Lancet, ii, 246-250).
The CP of Group B meningococci and of E. coli K1 are identical (poly xcex1(2xe2x86x928) NeuNAc) and serve as essential virulence factors and protective antigens for both pathogens (Grados, O., and Ewing, W. H. (1970) J. Infect. Dis. 122, 100-103; Kasper, D. L. et al. (1973) J. Immunol. 110, 262-268; Bhattacharjee, A. K. et al. (1975) J. Biol. Chem. 250, 1926-1932; Robbins, J. B. et al. (1974) N. Eng. J. Med. 290, 1216-1220). Poly xcex1(2xe2x86x928) NeuNAc is also a surface antigen of Moraxella nonliquefaciens and Pasteurella haemolytica, serotype A-2 (Bxc3x8vre, K. et al. (1983) NIHP Annals. 6, 65-73; Devi, S. J. N. et al. (1991) Infect. Immun. 59, 732-736; Adlam, C. et al. (1987) FEMS Microbiol. Lett. 42, 23-25). The latter is the major cause of outbreaks of pasteurellosis in young lambs which suggests that poly xcex1(2xe2x86x928) NeuNAc may serve as a virulence factor for yet another bacterial species.
Attempts to induce protective immunity to group B meningococci and E. coli K1 have been thwarted because poly xcex1(2xe2x86x928) NeuNAc, alone or complexed to outer membrane proteins, induced low and transient levels of IgM antibodies (Kasper, D. L. et al. (1973) J. Immunol. 110, 262-268; Wyle, F. A. et al. (1972) J. Infect. Dis. 126, 514-522; Zollinger, W. D. et al. (1979) J. Clin. Invest. 63, 836-842; Moreno, C. et al. (1985) Infect. Immun. 47, 527-533; Frasch, C. E. et al. (1988) J. Infect. Dis. 158, 710-718; Lifely, M. R. et al. (1991) Vaccine 9, 60-66). Covalent attachment of periodate-treated (Jennings, H. and Lugowski, C. (1981) J. Immunol. 127, 1011-1018) or acid-hydrolyzed poly xcex1(2xe2x86x928) NeuNAc (Porro, M. et al. (1983) Med. Trop. 43, 129-132) to a protein also failed to elicit antibodies to this antigen. Further, this CP has been considered as a xe2x80x9cself antigenxe2x80x9d, because xcex1(2xe2x86x928) NeuNAc is found as monomers or dimers on glycoproteins and gangliosides in adults and up to ≈11 residues in fetal tissues including N-CAMs (Finne, J. et al. (1983) Lancet, ii, 355-357; Finne, J. et al. (1987) J. Immunol. 138, 4402-4407; Soderstrom, T. et al. (1984) N. Eng. J. Med. 310, 726-727). Accordingly, investigators have studied other components, such as LPS, outer membrane proteins and iron-binding proteins, or chemically modified poly xcex1(2xe2x86x928) NeuNAc, as potential vaccines (Zollinger, W. D. et al. (1979) J. Clin. Invest. 63, 836-842; Moreno, C. et al. (1985) Infect. Immun. 47, 527-533; Frasch, C. E. et al. (1988) J. Infect. Dis. 158, 710-718; Jennings, H. J. et al. (1984) Infect. Immun. 43, 407-412; Jennings, H. J. et al. (1986) J. Immunol. 137, 1708-1713; Frasch, C. E. (1989) Clin. Microbiol. Rev. 2(Suppl), S134-S138).
Most newborns and adults have bactericidal antibodies to the three major serogroups (A,B,C) of meningococci (Goldschneider, I. et al. (1969) J. Exp. Med. 129, 1307-1326); most of the bactericidal activity, including of group B meningococci, was removed by adsorption with the homologous CP (Frasch, C. E. et al. (1988) J. Infect. Dis. 158, 710-718; Brandt, B. L. et al. (1972) J. Immunol. 108, 913-920; Kasper, D. L. et al. (1973) J. Infect. Dis. 127, 378-387; Skevakis, L. et al. (1984) J. Infect. Dis. 149, 387-396). The peak incidence of disease caused by meningococci, including group B, is when the maternally-derived antibodies have waned and the adult levels have not yet developed (Gotschlich, E. C. (1984) in Bacterial Vaccines. Ed. Germanier (Academic Press, NY) pp. 237-255; Goldschneider, I. et al. (1969) J. Exp. Med. 129, 1307-1326). Rises in poly xcex1a(2xe2x86x928) NeuNAc antibodies, including those of the IgG isotype, are detectable in patients convalescent from group B meningococcal meningitis (Wyle, F. A. et al. (1972) J. Infect. Dis. 126, 514-522; Zollinger, W. D. et al. (1979) J. Clin. Invest. 63, 836-842; Frasch, C. E. et al. (1988) J. Infect. Dis. 158, 710-718; Skevakis, L. et al. (1984) J. Infect. Dis. 149, 387-396; Craven, D. E. et al. (1982) Infect. Immun. 37, 132-137; Mandrell, R. E. and Zollinger, W. D. (1982) J. Immunol. 129, 2172-2178; Leinonen, M. and Frasch, C. E. (1982) Infect. Immun. 38, 1203-1207). Polyclonal and monoclonal (mAb) poly xcex1(2xe2x86x928) NeuNAc antibodies were raised in animals by multiple intravenous injections of bacteria (Robbins, J. B. et al. (1974) N. Eng. J. Med. 290, 1216-1220; Moreno, C. et al. (1985) Infect. Immun. 47, 527-533; Mandrell, R. E. and Zollinger, W. D. (1982) J. Immunol. 129, 2172-2178; Allen, P. Z. et al. (1982) J. Clin. Microbiol. 15, 324-329; Craven, D. E. et al. (1979) J. Clin. Microbiol. 10, 302-307; Frosch, M. et al. (1985) Proc. Natl. Acad. Sci. (USA) 82, 1194-1198). Monoclonal antibodies to this antigen were identified in a healthy 81 year old male and from hybridoma cultures (Kabat, E. A. et al. (1986) J. Exp. Med. 164, 642-654; Kabat, E. A. et al. (1988) J. Exp. Med. 168, 699-711; Raff, H. V. et al. (1988) J. Infect. Dis. 157, 118-126). These antibodies exert biologic activities which have been correlated with protective immunity; 1) complement-dependent bacteriolysis on Group B meningococci (Gotschlich, E. C. (1984) in Bacterial Vaccines. Ed. Germanier (Academic Press, NY) pp. 237-255; Goldschneider, I. et al. (1969) J. Exp. Med. 129, 1307-1326); 2) protection against lethal infection of rodents by E. coli K1 (Robbins, J. B. et al. (1974) N. Eng. J. Med. 290, 1216-1220; Glode, M. P. et al. (1977) Infect. Immun. 16, 75-80; Kim, K. S. et al. (1985) Infect. Immun. 50, 734-737).
There are two other bacterial NeuNAc polymers: 1) group C N. meningitidis CP composed of poly xcex1(2xe2x86x929) NeuNAc; most strains are variably O-acetylated at C7 or C8 (Bhattacharjee, A. K. et al. (1975) J. Biol. Chem. 250, 1926-1932). Although differing from poly xcex1(2xe2x86x928) NeuNAc only by linkage, poly xcex1(2xe2x86x929) NeuNAc is immunogenic and is a licensed vaccine against group C meningococci (World Health Organization Expert Committee on Biological Standardization. (1977) Technical Report Series, 610. WHO, Geneva, Switzerland); 2) E. coli K92 CP (FIG. 1) with the disaccharide repeat unit of alternating xcex1(2xe2x86x928),xcex1(2xe2x86x929) NeuNAc (The structure of this polysaccharide can be written as 9)-NeuNAc-xcex1-(2xe2x86x928)-NeuNAc-xcex1-(2xe2x86x92.) (Robbins, J. B. et al. (1972) Infect. Immun. 6, 651-656; Glode, M. P. et al. (1977) J. Infect. Dis. 135, 94-102; Egan, W. et al. (1977) Biochem. (USA) 16, 3687-3692; Glode, M. P. et al. (1979) J. Infect. Dis. 139, 52-59). Both group B and group C meningococcal antisera precipitate with E. coli K92 CP (Glode, M. P. et al. (1977) J. Infect. Dis. 135, 94-102; Egan, W. et al. (1977) Biochem. (USA) 16, 3687-3692; Glode, M. P. et al. (1979) J. Infect. Dis. 139, 52-59). Multiple i.v. injections of killed E. coli K92 bacteria induced precipitating antibodies to poly xcex1(2xe2x86x929) NeuNAc and to poly xcex1(2xe2x86x928),xcex1(2xe2x86x929) NeuNAc but not to poly xcex1(2xe2x86x928) NeuNAc (Glode, M. P. et al. (1977) J. Infect. Dis. 135, 94-102). Injection of E. coli K92 CP induced poly xcex1(2xe2x86x929) NeuNAc antibodies in adult volunteers; antibodies to poly xcex1(2xe2x86x928) NeuNAc were not measured (Glode, M. P. et al. (1979) J. Infect. Dis. 139, 52-59).
It is a general object of this invention to provide a polysaccharide-protein conjugate and a vaccine.
It is a specific object of this invention to provide a polysaccharide-protein conjugate capable of eliciting serum IgG and IgM antibodies to poly xcex1(2xe2x86x928) NeuNAc, or to both poly xcex1(2xe2x86x928) NeuNAc and poly xcex1(2xe2x86x929) NeuNAc, or to poly xcex1(2xe2x86x928),xcex1(2xe2x86x929) NeuNAc.
It is a further object of this invention to provide a pharmaceutical composition suitable for use in preventing systemic infections.
It is another object of this invention to provide a method of preventing systemic infections.
It is a further object of this invention to provide a method of preventing systemic infections caused by Groups A, B, and C Neisseria meningitidis. 
It is another object of this invention to provide a method of producing a polysaccharide-protein conjugate.
Further objects and advantages of the present invention will be clear from the description that follows.
In one embodiment, the present invention relates to a polysaccharide-protein conjugate comprising a polysaccharide and a carrier protein wherein the conjugate is capable of eliciting serum IgG and IgM antibodies to poly xcex1(2xe2x86x928) NeuNAc, or to both poly xcex1(2xe2x86x928) NeuNAc and poly xcex1(2xe2x86x929) NeuNAc, or to poly xcex1(2xe2x86x928),xcex1(2xe2x86x929) NeuNAc in a mammal or bird.
In another embodiment, the present invention relates to a pharmaceutical composition and a vaccine comprising a polysaccharide-protein conjugate in an amount sufficient to prevent systemic infections, and a pharmaceutically acceptable diluent, carrier, or excipient.
In a further embodiment, the present invention relates to a method of preventing systemic infections in an animal comprising administering to the animal an amount of a polysaccharide-protein conjugate sufficient to effect the prevention.
In another embodiment, the present invention relates to a method of preventing systemic infections caused by Groups A,-B, and C Neisseria meningitidis in an animal comprising administering to the animal the above-described polysaccharide-protein conjugate and a Group A meninococcal polysaccharide-protein conjugate under conditions such that the infections are prevented.
In yet another embodiment, the present invention relates to a method of producing a polysaccharide-protein conjugate comprising derivatizing a polysaccharide and conjugating the derivatized polysaccharide to a protein.